Gaps and inconsistencies in the current knowledge and implementation of biosafety and biosecurity practices for rickettsial pathogens

Introduction Rickettsia spp. and Orientia spp. are the causes of neglected infections that can lead to severe febrile and systemic illnesses in humans. Implementing proper biosafety practices when handling these pathogens is crucial to ensure a safe and sustainable work environment. It is essential to assess the current knowledge and identify any potential gaps to develop effective measures that minimise the risk of exposure to these pathogens. By doing so, we can establish a comprehensive framework that promotes safety, mitigates hazards, and safeguards the well-being of personnel and the surrounding community. Methods and results This review aimed to synthesise and determine the evidence base for biosafety precautions for Rickettsia spp. and Orientia spp. pathogens. Enhancing our understanding of the relative infectious risk associated with different strains of Rickettsia and Orientia spp. requires identifying the infectious dose of these pathogens that can cause human disease. The application of risk groups for Rickettsia and Orientia spp. is inconsistent across jurisdictions. There is also incomplete evidence regarding decontamination methods for these pathogens. With regards to Orientia spp. most of the available information is derived from experiments conducted with Rickettsia spp. Conclusions Rickettsia and Orientia spp. are neglected diseases, as demonstrated by the lack of evidence-based and specific biosafety information about these pathogens. In the case of Orientia spp., most of the available information is derived from Rickettsia spp., which may not be appropriate and overstate the risks of working with this pathogen. The advent of effective antibiotic therapy and a better understanding of the true hazards and risks associated with pathogen manipulation should inform decisions, allowing a sustainable and safe work environment. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-024-09151-0.


Introduction
The World Health Organization (WHO) released the fourth edition of the Laboratory Biosafety Manual (LBM4) in 2020 [1], which advocates for risk-based biosafety using established knowledge.The WHO LBM4 emphasizes risk-based biosafety to improve laboratory biological risk management, particularly in low-resource settings [2].This review discusses the general characteristics, biosafety evidence, and other important information regarding Rickettsia and Orientia spp.pathogens.These pathogens cause scrub typhus, typhus group, and spotted fever group rickettsiosis.Our review also highlights gaps in the current evidence and regulatory inconsistencies and provides recommendations for sustainable risk-based biosafety practices while working with Rickettsia and Orientia spp.pathogens using the principles promoted by the WHO.

Characteristics
The family Rickettsiaceae, order Rickettsiales, class Alphaproteobacteria, and phylum Proteobacteria are obligate intracellular Gram-negative bacteria, including the Rickettsia and Orientia genera [3].The largest genus, Rickettsia, is divided into three antigenic subgroups responsible for several highly virulent diseases, including spotted fever (SFG) and typhus groups (TG), and a transitional group (TRG) comprising R. australis, R. felis and R. akari [4,5].The Orientia genus comprises the scrub typhus group (STG).There are more than 30 species of SFG rickettsiae, including the prototype R. rickettsii (causing Rocky Mountain spotted fever or RMSF) and other prominent members, including R. conorii (causing Mediterranean spotted fever or MSF) and R. honei (causing Flinders Island spotted fever).Regarding the TG, the two members are R. typhi, the causative agent of murine typhus, and R. prowazekii, the cause of epidemic typhus [6,7].Orientia tsutsugamushi, the STG prototype, causes scrub typhus and has several distinct antigenic types, including Karp, Kato and Gilliam [8]; however, more than 20 types of Orientia spp.have been identified.

Clinical disease
Scrub typhus, typhus group and spotted fever group rickettsiosis have similar signs and symptoms, including fever, headache, rash, and muscle pain [7].An eschar, a key pathognomonic sign of STG and SFG rickettsiosis, may sometimes be present as a dark scab-like lesion and can be found at the site of the mite or tick bite [7].

Treatment
Rickettsial diseases can be effectively treated with the tetracycline class of antibiotics.Doxycycline is the first choice due to its high efficacy [5,15].However, in the late 1990s, there was suspicion of doxycycline-resistant strains of O. tsutsugamushi following the observation of increased fever clearance times in a small number of scrub typhus patients in Chiangrai, northern Thailand [16].Subsequently, studies have demonstrated that doxycycline-resistant scrub typhus is a misconception, with treatment outcomes likely to be determined by other bacterial, host, and pharmacological factors [17].Azithromycin may be considered an alternative for scrub typhus treatment [15] but not murine typhus [18].

Risk group classification and biosafety levels
Orientia tsutsugamushi and most of Rickettsia spp.are classified as risk group (RG) 3 pathogens in the United States, United Kingdom, Australia, Singapore, Germany, Switzerland, Belgium, and the European Union [19,20].However, there are inconsistencies in the designation of RGs depending on the pathogen and the country.Table 1 summarizes global RG classifications for O. tsutsugamushi and selected Rickettsia spp.

Infectious dose
The infectious dose required to cause human infection for Rickettsia and Orientia pathogens remains insufficiently understood; however, for some rickettsiae, there are results from animal and in vitro models that can provide useful information.Using modelling, the estimated infectious dose for R. rickettsii is 23 ID 50 (50% infectious dose), with a 95% confidence interval of 1 to 89 organisms [21] having used results from studies in non-human primates [22][23][24], and humans [25] (summarized study results presented in Table ;2).In vitro and in vivo studies have also been performed for other Rickettsia spp. in animal and in vitro models also using results from previous studies for R. prowazekii [26] (Cairo 3 strain), R.typhi [26], R. canada (now known as R. canadensis) [27], R. rickettsii [28], R. conorii [28] and R. sibirica [29] (Table ;2).Infectious dose results demonstrated differences in the susceptibility of the animal species [30], with guinea pigs (GP) generally more susceptible than mice (M), with minimum ID 50 of 393 and 15 (M) and 1 and 8 (GP) for R. prowazekii (Breinl and Cairo 3 strains); 2 (M) and 5 (GP) for R. typhi; 9.7 × 10 4 (M) and 1.1 × 10 4 (GP) for R. canada; 1.1 × 10 4 (M) and 126 (GP) for R. rickettsii (Sheila Smith) and 1.6 × 10 4 (M) and 21(GP) for R. rickettsii (R strain) [30] (Table ;2).The infectious dose for Orientia spp.has not been determined, however, mouse [31] and hybrid models with non-human primates [32] have been developed for vaccine assessment purposes.It has been shown that there are differences in the severity of illness caused by various strains of O. tsutsugamushi in animals [33,34]..

Biocontainment and personal protective equipment (PPE)
When assessing the level of risk involved in handling rickettsial pathogens, it is imperative to take into account both the nature of the pathogen and the type of procedure that will be performed.Factors such as pathogenicity, virulence, and the likelihood of aerosolization must be considered.Additionally, the specific activity to be performed (e.g., serology, inoculation of specimens into culture, animal experiments, initial in vitro growth of an isolate, or large-scale production) must also be taken into consideration as the risk will vary depending on the activity.Guidance is provided on these matters by the US [35], Canada [36], UK [37], Australia [38], Singapore [39], Switzerland [40] and Thailand [41] regarding recommendations for biocontainment requirements and special practices and procedures when working with rickettsial pathogens (see Table 3 for full details).In these jurisdictions, the guidance recommends that non-propagative work with clinical specimens be performed in a  Biosafety Level (BSL) 2 facility using BSL2 practices for NAATs and serology testing.Canada, the UK, Australia, and Singapore require all in vitro propagation of the rickettsial pathogens to be performed at BSL3 containment.In the US, procedures involving the in vitro propagation of rickettsial pathogens are mandated to use "BSL3 practices and containment equipment… for activities involving culture propagation or specimen preparation and propagation of clinical isolates known to contain or potentially containing Rickettsia spp.pathogenic to humans.".However, somewhat confusingly, the US regulations also state, "BSL2 facilities with BSL3 practices are recommended for all manipulations of known or potentially infectious materials, including… and inoculation, incubation, and harvesting of embryonated eggs or cell cultures." The US has specific guidance for working with Orientia spp.pathogens [35] in the BMBL 6th edition; however, locations such as Australia, where scrub typhus is endemic (northern Queensland, Northern Territory) does not have specific guidance [38].In vivo animal work with rickettsial pathogens is normally confined to BSL-3.However, recently, a mouse model for acute lethal rickettsial disease, using R. parkeri Atlantic Rainforest strain and C3H/HeN mice, has been described with the advantage that this model can be studied in an animal BSL2 containment level [42].

Select Agent requirements
In

Post-exposure prophylaxis
Post-exposure prophylaxis (PEP) following a laboratory or clinical exposure to a rickettsial pathogen is potentially lifesaving, depending on the pathogen and the route of inoculation.
Guidance is provided regarding PEP following a laboratory incident, although it is confined to RMSF exposures.The Canadian guidance [10] specifies 100 mg of doxycycline taken twice daily for 5-7 days and until the patient is afebrile for at least 2-3 days (Table S1).The US guidance provided by CDC/NIH [35] detailed in Table S1 does not specify the anti-rickettsial chemotherapy required; however, it also recommends infrastructure development such as availability of an experienced medical officer, signs and symptoms of disease training, nonpunitive, anonymous reporting system for accidents; and the reporting of all febrile illnesses.

Laboratory-acquired infections
There are numerous reports of laboratory-acquired infections (LAIs) caused by rickettsial pathogens.RMSF has been acquired through needlesticks [43,44] (Table S1) and aerosol exposures [45], centrifugation errors, equipment failure, the blending of infected ticks, and direct tick bites when working in the field.Before the introduction of biosafety practices (i.e., biological safety cabinets and PPE) and the use of antibiotics for PEP (i.e., before 1940), there were 63 laboratory-related infections recorded, including 11 deaths [10,46].Whilst there are some discrepancies in the absolute numbers, many of these LAIs were summarised by Pike [47], where RMSF (n = 13), scrub typhus (n = 7) and epidemic typhus (R. prowazekii) (n = 3) fatalities were described.In the same article, the infections and subsequent deaths [47] of Howard Ricketts and S. J. M. von Prowazek due to infections acquired during research activities were eponymously honoured due to their dedication and sacrifice.Orientia tsutsugamushi and R. typhi LAIs were often the result of aerosol exposures and needlesticks, often without any risk mitigation for aerosol exposure or animal handling [48].A study aggregating LAI infections in O. tsutsugamushi and R. typhi reported that between 1931 and 2000, 25 scrub typhus LAIs caused eight deaths and 35 murine typhus infections with no deaths [48], with the cause being largely aerosol and self-inoculation-related [48].Notably, all O. tsutsugamushi LAIs fatalities occurred exclusively during the pre-antibiotic era [48], demonstrating the effectiveness of the tetracycline group PEP.Following the introduction of antibiotic treatments, later reviews recorded ten reports of LAI R. typhi between 1941 and 1995, resulting in 35 laboratory-borne infections and no fatal cases distributed in countries such as the US, UK, South Korea, Malaysia, and Switzerland [48].

Disinfection and decontamination
Inactivation of rickettsial pathogens normally relies on the use of chemical disinfectants.However, there is often a lack of clear evidence; several reports and guidance documents recommend using chemicals for the empirical inactivation of rickettsial pathogens.Chemical inactivation includes alcohols (ethanol, isopropanol), aldehydes (formaldehyde, paraformaldehyde, glutaraldehyde), alkalis (sodium hydroxide, ammonium hydroxide), halogens (sodium hypochlorite, iodine), peroxygens (accelerated hydrogen peroxide, e.g., Rescue®), potassium peroxymonosulfate (Virkon-S®), peroxyacetic acid, (Oxy-Sept® 333), phenolic compounds (Lysol®, O-Syl®, Amphyl®, Tek-Trol®, Pheno-Tek II®), sodium dodecyl sulphate (Qiagen ATL); however, specific concentrations and durations of exposure vary and in many cases have not been validated (Table S1).Limited studies have been conducted to evaluate the efficacy of irradiation for inactivating rickettsiae;   S1).Combinations of chemicals and radiation have been used to inactivate O. tsutsugamushi; however, the justification for this combination was mandated by the requirement for rickettsial inactivation in blood products.Thermal treatment is effective for inactivating rickettsial pathogens.Exposure to dry heat at a temperature of 56 °C (Table S1) or humid heat at 121 °C was effective in inactivating most rickettsial pathogens, although there are variations in contact time depending on the pathogen (Table S1).

Discussion
The review has identified knowledge gaps related to biosafety issues concerning Orientia spp.and Rickettsia spp.pathogens.A summary of the evidence is presented in Table S1, and an overview of these gaps is presented in Table 4.

Differentiation of Orientia spp. and Rickettsia spp. biosafety aspects
The evidence presented in this review mainly focuses on Rickettsia spp., primarily RMSF and epidemic typhus, which are causes of serious clinical illness.Biosafety and  biocontainment measures have been recommended for Orientia spp.based on the characteristics of Rickettsia spp.However, it is not appropriate to extrapolate similarities between the two organisms regarding susceptibility to physical and chemical inactivation procedures.Such an approach is not comparing "like with like".Therefore, a "one size fits all" approach is used for Orientia spp.biosafety practices are not supported by clear evidence and bypass the principles of risk-based biosafety.

Inconsistencies in the classification of risk groups
Inconsistencies in applying risk group classifications for Orientia spp.and Rickettsia spp.have been observed across different jurisdictions, as evidenced in Table 1.
The classification of O. tsutsugamushi has been subject to debate, which has implications for the practical application of risk and mitigations, including biosafety levels.
Implementing risk-based biosafety, which considers both the pathogen being manipulated and the activity being performed, is strongly advocated [1,49].It has been proposed that Orientia spp.be reclassified as RG2 [19], given that pathogens in this group present a moderate risk to the individual but a low risk to the community, especially given that effective post-exposure prophylaxis is available in the case of laboratory exposures.While there is no argument that R. rickettsii and R. prowazekii should remain at RG3, we propose that consideration should be given to the reclassification of numerous non-RMSF SFG, TRG, R. typhi and Orientia spp. to RG2 pathogens.

Inconsistent recommendations for risk-based biosafety and containment levels
Using a one-size-fits-all approach for in vitro growth of Orientia spp.and Rickettsia spp.pathogens, such as mandatory BSL3-type containment facilities, inhibits research and does not apply a risk-based approach.The lack of appropriate containment facilities for the culture of rickettsial pathogens was recently recognised in a review of rickettsial diseases in India [50] and Europe [51].Using a liberal interpretation of the somewhat inconsistent US regulations, implementing BSL3 practices in BSL2 containment laboratory facilities is likely to be sufficient to maintain a safe workplace depending on the scale of the activities with only high-risk pathogens (i.e., R. rickettsii and R. prowazekii) or large-scale propagation requiring BSL3 practices and containment facilities.The WHO LBM4 [1] espouses a biosafety approach that comprehensively evaluates risk, enabling customized control measures for laboratory procedures.Blacksell et al. [19] proposed a three-tiered risk classification system (low, medium, and high), facilitating a sustainable and secure work environment while optimizing limited resources.They recommended that low-risk activities involving Orientia spp.(i.e., NAATs and serology using clinical specimens) be conducted inside a biological safety cabinet within a BSL2 containment laboratory while employing standard personal protective equipment [19].This is crucial because most rickettsial illnesses, particularly scrub typhus, manifest in low-resource settings with limited access to high containment facilities.It is possible to safely isolate representative pathogens in small to medium-scale (i.e., low to medium-risk) in vitro experiments to characterize and produce diagnostic reagents.This can be achieved by implementing BSL3 practices in a BSL2 containment laboratory, eliminating the need for high containment facilities and enabling investment in good laboratory practices and procedures for staff.Large-scale culture of Orientia spp.and Rickettsia spp.would be considered high-risk and performed using BSL3 practices and containment laboratory facilities [19].

Infectious dose
The infectious dose associated with individual pathogens may differ considerably and contribute significantly to the risk profile and the associated hazards (i.e., low infectious dose = high infectious risk).Accurate determination of the infectious dose for Rickettsia and Orientia spp.would provide a better understanding of the relative

Infectious dose
The infectious dose of Rickettsia and Orientia spp. is considered low but has not been determined.

Classification of risk groups
Debate regarding the risk group classification of O. tsutsugamushi -Blacksell et al. [19] advocated for the reclassification of Orientia spp. to RG2 from RG3 based on the fact that pathogens in RG2 pose a moderate risk to the individual but a low risk to the community.Furthermore, there is effective treatment available.
-RG impacts the practical application of risk, and mitigations, including biosafety levels.The application of risk-based biosafety that considers pathogen and the activity being performed rather than a "one size fits all" approach to risk mitigation and control strategies.o effectiveness and optimal conditions for gaseous decontamination of Rickettsia and Orientia spp.
risk-associated strains and inform the accurate determination of pathogen risk groups; however, the existing infectious dose information does provide general guidance.Without a complete understanding of the infectious dose required to cause human infections, or that of surrogate animal models, via various routes, it is impossible to accurately determine the true risks associated with manipulating the pathogen [52][53][54].

Disinfection and decontamination
The majority of rickettsial pathogens are considered labile microorganisms [55].Rickettsia spp.can synthesize lipopolysaccharide (LPS), whereas O. tsutsugamushi lacks this ability [56].Rickettsia spp.can synthesise a complete peptidoglycan cell wall; however, O. tsutsugamushi only possesses a subset of peptidoglycan biosynthesis genes, which generates a less robust peptidoglycan-like structure [56,57], making it more susceptible to inactivation.The cell membrane of Orientia spp.has characteristics that make it more susceptible to chemical and physical inactivation methods than other bacteria classified at lower-risk group levels.There is insufficient evidence to determine the most effective decontamination methods for Rickettsia and Orientia spp.

Conclusions
Rickettsia and Orientia spp.are neglected diseases, as demonstrated by the lack of evidence-based and specific biosafety information about these pathogens.In the case of Orientia spp., most of the available information is derived from Rickettsia spp., which may not be appropriate and overstate the risks of working with this pathogen.The advent of effective antibiotic therapy and a better understanding of the true hazards and risks associated with pathogen manipulation should inform decisions, allowing a sustainable and safe work environment.

Table 2
Institutes of Health, USA BMBL-Biosafety in Microbiology and Biomedical Laboratories, 6th ed ACDP -Advisory Committee on Dangerous Pathogens, 5th ed FSPII-First Schedule Part II Summary of results of studies investigating minimum infectious doses of rickettsiae in animal and in vitro models PFU -plaque forming units ID 50 − 50% infectious doses FD 50 − 50% fever dose per gram of yolk sac a Numbers of rickettsiae that constitute 1 plaque forming units (PFUs) or 1 50% effective dose in terms of cytopathic degeneration, infection, or fever b Will seroconvert 50% of the exposed mice c Minimum amount in organisms required to induce mortality or morbidity in at least one Rhesus monkey test group following aerosol exposure Claims lack clear supporting evidence and recommendations for chemical disinfectants often lack specific working concentrations and contact times.Validation methods often do not state the pathogen concentration, which can influence the chemical concentration or contact time required to kill the pathogen completely.Peroxygen-based disinfectants (Virkon®, Rescue®, etc.) are widely used in clinical and research labs to inactivate Rickettsia and Orientia spp.lack evidence.Evidence for inactivating Rickettsia spp.by irradiation is limited.Optimal heat and irradiation conditions for Orientia spp.are unknown.No evidence exists for gaseous decontamination of Rickettsia and Orientia spp.

Table 1
Summary of risk group designation for Rickettsia spp.andOrientia spp.based on the ABSA international database[20]

Table 3
Biocontainment and mitigation regulations for Rickettsia and Orientia spp however, some methodologies are reportedly successful (Table Where larger volumes or very high concentrations of the microorganisms are to be handled, the risk of infection or inadvertent release from containment can be higher and additional precautions or an increase in physical containment level may be appropriate"

Table 4
Summary of gaps in biosafety evidence for Rickettsia spp.and Orientia spp